The U.S. Centers for Disease Control and Prevention (CDC) defines a bioterrorism attack as the deliberate release of viruses, bacteria, or other germs (agents) used to cause illness or death in people, animals, or plants. According to the CDC, these agents are typically found in nature, but it is possible that they could be changed to increase their ability to cause disease, make them resistant to current medicines, or to increase their ability to be spread into the environment. The CDC warns that these biological agents can be spread through the air, through water, or in food, and that terrorists may use biological agents because they can be extremely difficult to detect and do not cause illness for several hours to several days.
The 2001 anthrax attacks are the most recent bioterrorism attack in the United States. Since these attacks, the United States and the Animal and Plant Health Inspection Services, the CDC, and the National Institutes of Allergy and Infectious Disease have initiated numerous research-based programs designed to better understand the pathogenic mechanisms of many bacteria and viruses that are capable of causing significant and highly problematic human disease and death. The U.S. Department of Health and Human Services or the U.S. Department of Agriculture has declared bio-agents that potentially pose a severe threat to public health and safety as select agents, and categorizes these select agents as Category A, B or C according their ease of dissemination and mortality and morbidity rates, among other factors.
Given the relative ease of genetic manipulation in bacterial pathogens based on technology developed in recent years, coupled with the dramatic increase in antibiotic resistance in virtually all bacterial pathogens, it is a distinct possibility that skilled scientists motivated to trigger a bioterrorist attack would be likely to engineer bacterial pathogen strains that are resistant to all current antibiotic regimens. Upon release and contamination, genetically engineered bacterial strains must be characterized and scientists will urgently have to screen novel chemical libraries for compounds that could kill these new antibiotic resistant strains without being toxic themselves to human cells, and in a time frame that prevents catastrophic population decimation.
There are many well-characterized libraries of chemical compounds with proven safety profiles already in the United States that could be screened for antibiotic efficacy against potentially new antibiotic-resistant strains of bacterial pathogens. However, in order to achieve the time-effective high throughput screening that would be necessary to provide a rapid response to intentional contamination, a platform providing a simple read-out for death and/or growth inhibition of these highly virulent bacterial pathogens is most desirable.
Simple read-out technologies are known in the art. For example, one such simple read-out based on death and/or growth inhibition is accomplished through the use bacterial pathogens and surrogates that have been genetically engineered to incorporate fluorescent protein reporters. As long as these genetically engineered bacterial pathogens are alive, the organism fluoresces, yielding a visible signal. Upon the death of the genetically engineered pathogen, the organism no longer fluoresces. Although such technology is known, it has not previously been considered in the development of counter-bioterrorism technology.
However, for the fluorescent protein reporters to be useful for high-throughput screening of large libraries of candidate compounds in the context of mounting a counter-bioterrorism response, the fluorescent measurements must require extremely short exposure time, as the cumulative exposure time of large libraries should be kept to a minimum. Ideally, less than 200 ms exposure time should be required to provide a salient read-out signal. The present inventors are unaware of existing fluorescent protein reporters at the single copy level in bacterial pathogen select agents that can meet such a short exposure requirement.
As such, there is a need for bacterial pathogens comprising fluorescent transcriptional fusion reporters capable of measurable signaling in a time frame suitable for rapid identification of an anti-bacterial agent. More effective methods for providing a rapid response to a bacterial contamination resulting from, for example, bioterrorism are also needed. In particular, there is a need for methods of screening candidate compounds for anti-bacterial efficacy useful for countering intentional contamination by altered bacterial pathogens resistant to existing antibacterial agents.